Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterised by autoantibody production and immune complex (IC) formation. Common autoantibodies are rheumatoid factor (RF) and those against citrullinated peptides (CCPs) [1]. Approximately 70% of all RA patients display rheumatoid factor and/or anti-CCP antibodies, and the presence of anti-CCP antibodies can be detected in serum several years before disease debut [2]. Most autoantibodies are of the IgG isotype, which have the potential to activate Fc gamma receptors (FcγRs) on leukocytes, such as macrophages, neutrophils, dendritic cells and B cells. Cross-linking of FcγRs by IgG-ICs leads to cellular effector functions such as phagocytosis, antibody-dependent cellular toxicity and release of inflammatory cytokines.

Three different classes of FcγRs have been identified in humans so far; FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Furthermore, FcγRII and FcγRIII exist in two isoforms, a and b, which carry out divergent functions. FcγRI is a high affinity receptor that binds monomeric IgG as well as IgG-ICs, while FcγRII and FcγRIII are low affinity receptors that predominantly bind IgG-ICs. FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb are activating receptors. FcγRI and FcγRIIIa consist of an α-chain with three and two Ig-domains respectively, which is connected with a cytoplasmic signalling subunit, the γ-chain. The γ-chain is responsible for intracellular signalling via its immunoreceptor tyrosine based activation motif (ITAM). FcγRIIa is a single chain receptor that contains an ITAM-motif in the cytoplasmic tail. FcγRIIb is an inhibitory receptor that is structurally similar to FcγRIIa, but has an immunoreceptor tyrosine based inhibitory motif in the cytoplasmic domain. FcγRIIb has been shown to have an important negative regulatory function on Fc receptor activation [3].

The involvement of FcγRs in experimental arthritis has been thoroughly investigated, and it is now clear that activating FcγRs are essential for the development of disease. Thus, mice lacking the common γ-chain or FcγRIII are protected from collagen-induced arthritis (CIA) as well as other experimental models of arthritis [4–8]. Consequently, FcγRIIb deficiency in mice leads to increased susceptibility to CIA [9, 10]. These findings emphasize the importance of FcγRs in the pathogenesis of experimental arthritis, which may also be true for arthritis in humans. A reported gene polymorphism of FcγRIIIa has been correlated with RA [11–13] as this polymorphism changes the receptor affinity for different IgG-subclasses [14, 15]. The FcγRIIIA 158 V/F allele variant has been especially associated with the risk of developing RA [16], although conflicting data exist [17]. Recently, it was also reported that there is an association between rheumatoid factor and the FcγRIIIa 158 V/F allele in RA patients [18] and that a functional variant of FcγRIIb is associated with increased joint destruction in RA but not disease susceptibility [19]. Moreover, several studies have shown that the percentage of FcγRIII positive monocytes is increased in peripheral blood of RA patients [20, 21] and that the expression levels of FcγRI, FcγRII and FcγRIII on RA monocytes are increased compared to healthy individuals [22–24], while FcγRIIb expression is unaffected [25].

It has previously been hard to obtain knowledge about FcγR expression in healthy synovial tissue for comparison with FcγR expression in RA patients. Synovia from trauma patients or osteoarthritis patients has often been used as control material [26, 27] and only limited studies have been done with healthy synovium [28]. Therefore, we have in this study investigated the expression of the different FcγRs using synovial tissue from healthy volunteers in comparison with RA synovia. We were particularly interested in investigating expression of the inhibitory FcγRIIb as this receptor has not previously been studied due to the lack of a specific antibody against it. Here, by using a novel FcγRIIb specific antibody, GB3, we demonstrate a pronounced FcγRIIb expression in the synovial inflammation in RA, which is in sharp contrast to the lack or weak staining of FcγRIIb in healthy synovia. Additionally, we could detect FcγRII and FcγRIII, but not FcγRI, in healthy synovial tissue. In the RA synovia, the expression of all activating FcγRs was significantly increased, regardless of disease duration. The synovial FcγRI expression could be reduced by intraarticular glucocorticoid treatment.

These data demonstrate that the inflammation in RA synovium is characterised by a pronounced expression of the inhibitory FcγRIIb, which suggests it has a role in counteracting the effects of activating FcγRs in RA synovia. Specific FcγRIIb expression patterns in RA or healthy synovia have not previously been described; nor has the expression of activating FcγRs in healthy synovia been described fully. Synovium from trauma patients has been reported to express FcγRI, II and III, and the FcγRII and III expression has been shown to be significantly lower in these patients compared to RA patients, while their FcγRI expression does not differ from that of RA synovia [27]. This is in contrast to our findings where we could not identify any FcγRI expression in healthy synovium. Thus, it appears that FcγRI is expressed as a consequence of a general inflammation in the joint, as we also observed FcγRI expression in synovial tissue from osteoarthritis patients (data not shown), although not to the same extent as seen in RA patients. The fact that FcγRI is absent from healthy synovial tissue but present in RA synovium and significantly decreased by administration of glucocorticoids indicates that FcγRI has a significant inflammatory role in the pathogenesis of arthritis. This is interesting and in line with experimental studies where FcγRI deficiency in mice leads to a reduced uptake of IgG-ICs and to decreased IC-induced inflammation [36]. It has also been reported that up-regulation of FcγRI leads to increased cartilage destruction in arthritic mice [37]. Similar to FcγRI, FcγRIIb was absent from, or only weakly expressed in, healthy synovia, whereas RA synovium clearly expressed FcγRIIb. This indicates a need for FcγRIIb to control the stimulatory activity of the ITAM-containing receptors in the RA joint.

Although the majority of RA macrophages expressed FcγRIIb, it was also evident that some macrophages did not. This may point towards a small subpopulation of FcγRIIb negative macrophages that may have extraordinary inflammatory capacities. We also observed FcγRIIb expression in lymphocyte infiltrates of RA synovium, which further suggests that infiltrating B cells may also be regulated by this receptor. In contrast to the near absence of FcγRIIb expression in healthy synovia, we clearly observed positive FcγRII staining with the pan anti-FcγRII mAb, most likely as a result of the presence of the activating FcγRIIa in healthy synovia. FcγRIII is also expressed in healthy synovia, ready to bind ICs caught in the joint. In RA synovium both FcγRII and FcγRIII expression was significantly increased, as has also previously been observed [26–28]. However, no association with disease duration and the degree of FcγR expression in RA patients could be shown, as both early and late patient groups expressed similar amounts of FcγRs. This suggests that the FcγRs are important during the whole disease course and not only in the induction phase of RA.

The importance of FcγRII and FcγRIII in arthritis has also been emphasized in animal models of RA. Transgenic mice expressing human FcγRIIa [38] develop CIA much earlier than wild-type mice and normally arthritis resistant mice become susceptible to arthritis when expressing FcγRIIa [39]. Furthermore, the induction of CIA is dependent on FcγRIII and, in particular, FcγRIII positive macrophages [4, 40]. Studies of RA monocytes/macrophages have also stressed the significance of FcγRII and FcγRIII in disease pathogenesis. Thus, FcγRII and FcγRIII are up-regulated on peripheral blood monocytes and FcγRIII expression is also enhanced on synovial fluid macrophages [21, 22].

The importance of FcγRII in RA was also noted in this study by the decrease in FcγRII expression (albeit not significant) after glucorticoid treatment. The decrease in FcγRII as well as FcγRI expression after local glucocorticoid injection was not due to a reduced number of macrophages in the tissue, as no significant difference in CD163 expression was found after treatment. In agreement, a previous study, where the same patient material was included, also found that the amount of CD163 and CD68 positive cells were not affected by intraarticular glucocorticoid treatment [41]. This indicates that FcγRs are down regulated from the cell surface by glucocorticoid treatment, which may help to explain some of the improvement seen in RA patients upon treatment with it, in addition to its reported suppressive effects on cytokines [41]. These results are in line with an earlier report that indirectly showed that FcγR expression is decreased on peripheral blood monocytes from autoimmune hemolytic anemia patients after systemic administration of corticosteroid, measured by radiolabelled IgG-binding [42]. In a more recent paper, FcγRI and II were shown to be decreased on peripheral blood monocytes one month after systemic therapy with daily low glucocorticoid doses [22]. Recent studies with other anti-rheumatic drugs have demonstrated that methotrexate treatment could reduce the expression of FcγRI and IIa on peripheral blood monocytes from RA patients, while the expression of FcγRIII was unaffected [43]. We did not see a clear reduction in FcγRIII expression after glucocorticoid treatment, which might indicate that FcγRIII expression is hard to modify using anti-rheumatic drugs. It is difficult to speculate on how relevant the reduction of FcγRs is for the physiological outcome of glucocorticoid treatment, since patients receiving an intraarticular injection of glucocorticoids experience an almost instant effect, but positive anti-inflammatory effects in the joint are seen several weeks after treatment.

Macrophages are present in the synovial lining layer of healthy synovium and the amount of macrophages in joints of RA patients is correlated with disease activity [44–46]. We identified the presence of FcγRI, II, IIb and III on RA synovial macrophages and, in addition, expression of FcγRI, FcγRII and FcγRIII revealed by DAB staining was significantly correlated with the expression of the macrophage marker CD163. This indicates that macrophages are likely to be involved in the IC-mediated damage in RA via their FcγR expression and they may also be responsible for antigen presentation to T cells, as macrophages were observed in close proximity to CD3 positive T cells in the RA synovium. The FcγR-positive macrophages were often localised perivasculary together with CD3-positive T cells, most likely as a result of recent extravasation. It is possible that presentation of antigen taken up via FcγRs on the macrophages may activate T cells to secrete cytokines. This could, in turn, lead to further activation of the macrophages and, thus, production of inflammatory cytokines, resulting in a continuous inflammatory state in RA joints.

Our findings demonstrate that expression of the inhibitory FcγRIIb, as well as of the activating FcγRI, FcγRII and FcγRIII, is increased in RA synovium, regardless of disease duration. The importance of FcγRI and FcγRIIb in the synovial inflammation of RA patients is further highlighted by the fact that healthy synovia lack FcγRI expression and substantially lack FcγRIIb expression. Furthermore, anti-inflammatory drugs, such as glucocorticoids, suppress FcγRI expression after local administration in the joint. These results clearly point towards a central role for the FcγRs in the synovial inflammation of RA.